Electrochemical test strip cards that include an integral dessicant

ABSTRACT

Electrochemical test strip cards that can be singulated to produce electrochemical test strips are provided. The electrochemical test cards are made up of two or more electrochemical test strip precursors, where each precursor is characterized by the presence of a dry reagent housing reaction chamber bounded by opposing electrodes. In gaseous communication with each reaction chamber of the card is an integrated desiccant. Also provided are methods of using the subject electrochemical test strips cards, as well as kits that include the same. The subject test strips and cards find use in the detection/concentration determination of a number of different analytes, including glucose.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 09/746,116,filed on Dec. 20, 2000, now U.S. Pat. No. 6,558,528, the completedisclosure of which is incorporated herein by reference for allpurposes.

FIELD OF THE INVENTION

The field of this invention is analyte determination, particularlyelectrochemical analyte determination and more particularly theelectrochemical determination of blood analytes.

BACKGROUND

Analyte detection in physiological fluids, e.g., blood or blood derivedproducts, is of ever increasing importance to today's society. Analytedetection assays find use in a variety of applications, includingclinical laboratory testing, home testing, etc., where the results ofsuch testing play a prominent role in diagnosis and management in avariety of disease conditions. Analytes of interest include glucose fordiabetes management, cholesterol, and the like. In response to thisgrowing importance of analyte detection, a variety of analyte detectionprotocols and devices for both clinical and home use have beendeveloped.

One type of method that is employed for analyte detection is anelectrochemical method. In such methods, an aqueous liquid sample isplaced into a reaction zone in an electrochemical cell comprising twoelectrodes, i.e., a reference and working electrode, where theelectrodes have an impedance which renders them suitable foramperometric measurement. The component to be analyzed is allowed toreact directly with an electrode, or directly or indirectly with a redoxreagent to form an oxidizable (or reducible) substance in an amountcorresponding to the concentration of the component to be analyzed,i.e., analyte. The quantity of the oxidizable (or reducible) substancepresent is then estimated electrochemically and related to the amount ofanalyte present in the initial sample.

A problem faced by manufacturers and users of these types ofelectrochemical test strips is reagent degradation due to waterexposure. For example, when the reagent composition of such strips isexposed to normal environmental humidity, the response of the test stripcan change dramatically and therefore confound the results obtained withthe strip.

As such, there is continued interest in the identification of newelectrochemical strip configurations in which the reagent composition ofthe strip is protected from contact with environmental humidity. Ofparticular interest would be the development of a card from which aplurality of test strips could be singulated, where the reagentcomposition in each card is protected from water mediated degradation.

Relevant Literature

Patent documents of interest include: U.S. Pat. Nos. 5,708,247;5,942,102; 5,951,836; 5,972,199; 5,989,917; 5,997,817; 6,151,110;6,125,292; WO 97/18465; WO 97/27483 and EP 871 033.

SUMMARY OF THE INVENTION

Electrochemical test strip cards that can be singulated to produceelectrochemical test strips are provided. The electrochemical test cardsare made up of two or more electrochemical test strip precursors, whereeach precursor is characterized by the presence of a dry reagent housingreaction chamber bounded by opposing electrodes. In gaseouscommunication with each reaction chamber of the card is an integrateddesiccant. Also provided are methods of using the subjectelectrochemical test strips cards, as well as kits that include thesame. The subject test strips and cards find use in thedetection/concentration determination of a number of different analytes,including glucose.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic for the assembly of a first embodiment ofthe subject test strip cards.

FIG. 2 provides a schematic for the assembly of a second embodiment ofthe subject test strip cards.

FIG. 3 provides a schematic for the assembly of a third embodiment ofthe subject test strip cards.

FIG. 4 a provides an exploded view of a test strip card according to thesubject invention, while FIG. 4 b provides an exploded view of a teststrip that is singulated from the card shown in FIG. 4 a.

FIGS. 5 a to 5 c provide graphical results of the data obtained from theexperiments reported in Example I.

FIG. 6 a provides an exploded view of a test strip card according to thesubject invention, while FIG. 6 b provides an exploded view of a teststrip that is singulated from the card shown in FIG. 6 a.

FIG. 7 provides graphical results of the data obtained from theexperiments reported in Example II.

FIG. 8 a provides an exploded view of a test strip card according to thesubject invention, while FIG. 8 b provides an exploded view of a teststrip that is singulated from the card shown in FIG. 8 a.

FIG. 9 a provides an exploded view of a test strip card according to thesubject invention, while FIG. 9 b provides an exploded view of a teststrip that is singulated from the card shown in FIG. 9 a.

FIGS. 10 a to 10 b provide graphical results of the data obtained fromthe experiments reported in Example III.

FIG. 11 a provides an exploded view of another test strip card accordingto the subject invention, while FIG. 11 b provides an exploded view of atest strip that is singulated from the card shown in FIG. 11 a.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Electrochemical test strip cards that can be singulated to produceelectrochemical test strips are provided. The electrochemical test cardsare made up of two or more electrochemical test strip precursors, whereeach precursor is characterized by the presence of a dry reagent housingreaction chamber bounded by opposing electrodes. In gaseouscommunication with each reaction chamber of the card is an integrateddesiccant. Also provided are methods of using the subjectelectrochemical test strips cards, as well as kits that include thesame. The subject test strips and cards find use in thedetection/concentration determination of a number of different analytes,including glucose.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, singular referencesinclude the plural, unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs.

Electrochemical Test Cards

As summarized above, the subject invention provides electrochemical teststrip cards that can be singulated into electrochemical test strips.More specifically, the electrochemical test strip cards can be cut intotwo or more, i.e., a plurality of electrochemical test strips.Generally, the cards can be singulated or cut into from about 2 to 100,usually from about 5 to 50 and more usually from about 10 to 30individual test strips.

As such, the test strip cards are characterized in that they include aplurality of adjacent test strip precursors, where by plurality is meantat least 2, where the number of precursors in a given card generallyranges from about 2 to 100, usually from about 5 to 50 and more usuallyfrom about 10 to 30. The dimensions of the subject cards may vary, butgenerally the cards have a length ranging from about 2 cm to 50 cm,usually from about 3 cm to 30 cm and more usually from about 6 cm to 20cm and a width ranging from about 0.5 cm to 10 cm, usually from about 1cm to 8 cm and more usually from about 2 cm to 5 cm. Thus, the teststrips that can be cut from the cards generally have a length thatranges from about 0.5 cm to 10 cm, usually from about 1 cm to 8 cm andmore usually from about 2 cm to 5 cm and a width that ranges from about0.1 cm to 2.5 cm, usually from about 0.2 cm to 1.5 cm and more usuallyfrom about 0.5 cm to 1 cm.

Each precursor of the card is characterized by including at least areaction chamber which is bounded by opposing electrodes and houses adry reagent composition. These features of the subject precursors aredescribed in greater detail infra in terms of the test strips that canbe produced from the subject cards.

A feature of the subject invention is that an integrated desiccant foreach reaction chamber is present in the subject cards. By integrated ismeant that the desiccant is a component or integral feature of the card,e.g., it is a component that is incorporated into the card, a componentpresent in one or more of the materials making up the card, e.g., alaminated covering material, etc., and the like. As the cards contain adesiccant for each reaction chamber, typically they include a pluralityof desiccant materials so that an individual desiccant material ispresent for each reaction chamber. As such, the number of individualdesiccant materials present in the cards generally ranges from about 2to 100, usually from about 5 to 10 and more usually from about 10 to30—one for each reaction chamber present on the card.

A variety of different types of desiccant materials may be employed,where representative desiccant materials include solid materials, e.g.,beads and strips or blocks of desiccant material, etc. Each desiccantmaterial should have a capacity of at least about 0.5 mg water per test,usually at least about 1 mg water per test and more usually at leastabout 1.5 mg water per test. The capacity of the desiccant materialsemployed in the subject cards typically ranges from 0.5 mg water pertest to 10 mg water per test, usually from about 0.75 mg water per testto 5 mg water per test and more usually from about 1.0 mg water per testto 3 mg water per test. Representative materials that may be employed asdesiccants include, but are not limited to: mol sieve, silica gel,CaSO₄, CaO and the like. Incorporated into the desiccant material may bean indicator that provides a detectable single, e.g., color change, thatcan be used to determine the remaining capacity of the desiccant, e.g.,to determine whether or not a desiccant has reached capacity withrespect to the amount of water that it can sequester. Indicatorcompounds of interest include, but are not limited to: CoCl₂ and thelike.

The cards are further characterized in that, prior to singulation intoindividual strips, each reaction chamber of each precursor is in gaseouscommunication with a desiccant material present on the card. By gaseouscommunication is meant that at least water vapor present in the reactionchamber is freely able to move to the desiccant and be sequesteredthereby.

In many embodiments, the desiccant material is generally present in,i.e., housed in, a desiccant chamber which is part of the card, and inmany embodiments incorporated into each strip. The desiccant chambersmust be of sufficient volume to house the desiccant materials, where thevolume of the desiccant chambers generally ranges from about 0.0015 ccto 0.15 cc, usually from about 0.010 cc to 0.10 cc and more usually fromabout 0.015 cc to 0.08 cc. The configuration of the chamber may varyconsiderably and depends primarily on the dimensions of the materialwhich is housed in the desiccant chamber.

Generally, a channel or tube connects the reaction chamber of eachprecursor to a desiccant chamber so that the requisite gaseouscommunication between the desiccant and the reaction chamber isestablished. The tube or channel often has a smallest dimension rangingfrom about 0.002 cm to 0.05 cm, usually from about 0.005 cm to 0.05 cmand may have a length that ranges from about 0 cm to 3 cm, usually fromabout 0.02 cm to 1.5 cm and more usually from about 0.15 cm to 5 cm.

The configuration of each desiccant chamber with respect to eachreaction chamber with which it is in gaseous communication may vary. Incertain embodiments, the desiccant chamber is in gaseous communicationwith a reaction chamber that is present on the same precursor, such thatwhen the card is singulated, the resultant test strip has a reactionchamber that is still in gaseous communication with a desiccant materialin a desiccant chamber. In alternative embodiments, the desiccantchamber is in gaseous communication with a reaction chamber that ispresent on an adjacent precursor, e.g., either the right or leftprecursor to it, such that when the card is singulated, the resultanttest strip has a reaction chamber that is no longer in gaseouscommunication with a desiccant material.

In certain embodiments, the card is configured such that singulationresults in the production of an electrochemical test strip that hasfluid entry and exit channels leading into and out of the reactionchamber which provide for fluid communication between the reactionchamber and the external environment of the test strip, where no suchcommunication existed prior to singulation. In other words, the card isconfigured so that when a test strip is cut from an end of the card, thecutting or singulation process results in the production of fluidingress and egress channels between the reaction chamber and theexternal environment of the strip, so that fluid sample can beintroduced into the reaction chamber and gas can leave the reactionchamber.

The subject test strip cards are typically present in a moisture vaporbarrier material which provides for a moisture vapor impermeable barrierbetween the card and the external environment. The barrier material maybe laminated onto the card to provide for a tight seal. Any convenientmoisture vapor impermeable material may be employed, whererepresentative materials include, but are not limited to: polyethylene,polypropylene, polystyrene, polyethylene terepthalate, rubber, polymersof fluorinated and/or chlorinated ethylene monomers, copolymers offluorinated and/or chlorinated ethylene monomers,polymethylmethacrylate, films coated with silicon oxide and the like. Incertain embodiments the cards further include calibration information;identification information, etc., which may be present on the card inthe form of a scannable bar code, or other information storage means.

The design of the cards may be varied to provide for a number ofdifferent electrical contact configurations in test strips that areultimately singulated from the cards. Representative alternative contactconfigurations are provided in FIGS. 1 to 3, described in greater detailinfra.

Representative test strip card configurations are now further describedin terms of the figures. FIG. 4 a provides an exploded view of a teststrip card according to one embodiment of the subject invention, whileFIG. 4 b provides an exploded view of a test strip cut from the cardshown in FIG. 4 a. In FIG. 4 a, test strip 40 is a multi-layer structuremade up of top and bottom layers 41 a & 41 b (e.g., 3M 425, which is alamination of 0.0028″ Al foil and 0.0018″ acrylic PSA), top and bottomelectrode layers 42 a & 42 b (e.g., 0.005 clear PET, Au coat (bottom) &0.005″ clear PET, Pd coat top side, respectively), and middle spacerlayer 43 (e.g., 0.003″ PET, 0.001 acrylic PSA both sides). Spacer layer43 has a pattern that provides for a reaction chamber 44, a desiccantchamber 45, fluid ingress channel 46, a channel 47 connecting thereaction chamber to the desiccant chamber, and a venting channel, 48,attached to the desiccant chamber. Desiccant 47 a (e.g., 2.5 mg 4A molseive beads) is located in the desiccant chamber. Also present arecutouts 49 a & 49 b in the electrode layers that allow for clearance ofdesiccant materials thicker than the combined thickness of 42 a, 42 band 43 upon assembly of the card. These cutouts also create a stopjunction at the edge of the desiccant chamber so that only a definedamount of fluid can enter the reaction chamber and channels. In thisconfiguration of the card, singulation of the card into an individualtest strip opens the fluid ingress channel such that fluid communicationis established between the reaction chamber of the strip and both theexternal environment and the desiccant chamber. In addition, the card isconfigured such that singulation results in a strip in which thedesiccant chamber and ultimately the fluid channels and reaction chamberare vented to the external environment, so that fluid ingression canproceed without being impeded by air pressure build-up. The test stripcard of FIG. 4 a has a precut 40 a that provides guidance for the finalcut 40 b employed to singulate the cards into strips.

FIG. 6 a provides an exploded view of a modification of the card of FIG.4 a, where a desiccant block or tape 61 (e.g., 60% 4A mole sieve; 1-3%glycol in PETG; approx 0.2×0.15×0.025″) is present in the desiccantchamber 45. Also shown is a representative singulation cut 60 a. FIG. 6b shows the details of a singulated strip. In FIGS. 6 a and 6 b, thefluid ingression channel 46 and the vent 48 have been shortened for thepurposes of the experiment described in example II.

FIG. 8 a provides an exploded view of a modified version of the stripshown in FIG. 6 a. In FIG. 8 a, the top and electrode layers have beencombined into single layers 81 and 82, where single layers 81 and 82have stamped regions 83 and 84 to accommodate desiccant block 61.

FIG. 9 a provides an exploded view of a modification of the strip ofFIG. 4 a, where channel 46 and vent 48 have been shortened according toexample III.

Yet another embodiment of the subject cards can be seen in FIG. 3. InFIG. 3, the design shown in FIG. 4 a has been modified so that thereaction chamber 31 of each precursor is in gaseous communication with adesiccant chamber 33 present on the precursor adjacent to it. FIG. 11 aprovides an exploded view of a test strip card according to theembodiment of FIG. 3, while FIG. 11 b provides an exploded view of atest strip cut from the card shown in FIG. 11 a. In FIG. 11 a, teststrip card 50 is a multi-layer structure made up of top and bottomlayers 51 a & 51 b, top and bottom electrode layers 52 a & 52 b, andmiddle spacer layer 53. Spacer layer 53 has a pattern that provides fora reaction chamber 54, a desiccant chamber 55, fluid ingress channel 56,a channel 57 which, prior to singulation, connects the desiccant chamber55 to the reaction chamber of an adjacent test strip (not shown), and aventing channel, 58, attached to the reaction chamber 54. Desiccant 57 ais located in the desiccant chamber 55. Also present are cutouts 59 a &59 b in the electrode layers that allow for clearance of desiccantmaterials thicker than the combined thickness of 52 a, 52 b and 53 uponassembly of the card. These cutouts also create a stop junction at theedge of the desiccant chamber so that only a defined amount of fluid canenter the reaction chamber and channels. In this configuration of thecard, singulation of the card into an individual test strip opens thefluid ingress channel such that fluid communication is establishedbetween the reaction chamber of the strip and both the externalenvironment and the desiccant chamber. In addition, the card isconfigured such that singulation results in a strip in which thedesiccant chamber and ultimately the fluid channels and reaction chamberare independently vented to the external environment, so that fluidingression can proceed without being impeded by air pressure build-up.The test strip card 50 of FIG. 11 a has a precut 50 a that providesguidance for the final cut 50 b employed to singulate the cards intostrips. As can be seen from the figure, in this configurationsingulation of the card into an individual test strip opens the fluidingress and egress channels such that fluid communication is establishedbetween the reaction chamber of the strip and the external environment.In addition, the card is configured such that singulation results in astrip in which the reaction chamber is no longer in gaseouscommunication with the desiccant chamber.

ELECTROCHEMICAL TEST STRIPS

As indicated above, the electrochemical test strip cards of the subjectinvention can be singulated or cut into individual electrochemical teststrips. The subject electrochemical test strips include two opposingmetal electrodes separated by a thin spacer layer, where thesecomponents define a reaction chamber, i.e., area or zone, in which islocated a redox reagent system.

As indicated above, the working and reference electrodes are generallyconfigured in the form of elongated rectangular strips. Typically, thelength of the electrodes ranges from about 1.9 to 4.5 cm, usually fromabout 2.0 to 2.8 cm. The width of the electrodes ranges from about 0.38to 0.76 cm, usually from about 0.51 to 0.67 cm. The reference electrodestypically have a thickness ranging from about 10 to 100 nm and usuallyfrom about 10 to 20 nm.

The working and reference electrodes are further characterized in thatat least the surface of the electrodes that faces the reaction area inthe strip is a metal, where metals of interest include palladium, gold,platinum, silver, iridium, carbon, doped tin oxide, stainless steel andthe like. In many embodiments, the metal is gold or palladium. While inprinciple the entire electrode may be made of the metal, each of theelectrodes is generally made up of an inert support material on thesurface of which is present a thin layer of the metal component of theelectrode. In these more common embodiments, the thickness of the inertbacking material typically ranges from about 51 to 356 μm, usually fromabout 102 to 153 μm while the thickness of the metal layer typicallyranges from about 10 to 100 nm and usually from about 10 to 40 nm, e.g.a sputtered metal layer. Any convenient inert backing material may beemployed in the subject electrodes, where typically the material is arigid material that is capable of providing structural support to theelectrode and, in turn, the electrochemical test strip as a whole.Suitable materials that may be employed as the backing substrate includeplastics, e.g. PET, PETG, polyimide, polycarbonate, polystyrene,silicon, ceramic, glass, and the like.

A feature of the electrochemical test strips produced from the subjectcards is that the working and reference electrodes as described aboveface each other and are separated by only a short distance, such thatthe distance between the working and reference electrode in the reactionzone or area of the electrochemical test strip is extremely small. Thisminimal spacing of the working and reference electrodes in the subjecttest strips is a result of the presence of a thin spacer layerpositioned or sandwiched between the working and reference electrodes.The thickness of this spacer layer generally should be less than orequal to 500 μm, and usually ranges from about 102 to 153 μm. The spacerlayer is cut so as to provide a reaction zone or area with at least aninlet port into the reaction zone, and generally an outlet port out ofthe reaction zone as well, i.e., the ingress and egress channelsdescribed above. The spacer layer may have a circular reaction area cutwith side inlet and outlet vents or ports, or other configurations, e.g.square, triangular, rectangular, irregular shaped reaction areas, etc.The spacer layer may be fabricated from any convenient material, whererepresentative suitable materials include PET, PETG, polyimide,polycarbonate, and the like, where the surfaces of the spacer layer maybe treated so as to be adhesive with respect to their respectiveelectrodes and thereby maintain the structure of the electrochemicaltest strip. Of particular interest is the use of a die-cut double-sidedadhesive strip as the spacer layer.

The electrochemical test strips produced from the subject cards includea reaction chamber, zone or area that is defined by the workingelectrode, the reference electrode and the spacer layer, where theseelements are described above. Specifically, the working and referenceelectrodes define the top and bottom of the reaction area, while thespacer layer defines the walls of the reaction area. The volume of thereaction area is at least about 0.1 μL, usually at least about 1 μL andmore usually at least about 1.5 μL, where the volume may be as large as10 μL or larger. As mentioned above, the reaction area generallyincludes at least an inlet port, and in many embodiments also includesan outlet port. The cross-sectional area of the inlet and outlet portsmay vary as long as it is sufficiently large to provide an effectiveentrance or exit of fluid from the reaction area, but generally rangesfrom about 9×10⁻⁴ to 5×10⁻³ cm², usually from about 1.3×10⁻³ to 2.5×10⁻³cm².

Present in the reaction area is a redox reagent system, which reagentsystem provides for the species that is measured by the electrode andtherefore is used to derive the concentration of analyte in aphysiological sample. The redox reagent system present in the reactionarea typically includes at least an enzyme(s) and a mediator. In manyembodiments, the enzyme member(s) of the redox reagent system is anenzyme or plurality of enzymes that work in concert to oxidize theanalyte of interest. In other words, the enzyme component of the redoxreagent system is made up of a single analyte oxidizing enzyme or acollection of two or more enzymes that work in concert to oxidize theanalyte of interest. Enzymes of interest include oxidases,dehydrogenases, lipases, kinases, diphorases, quinoproteins, and thelike.

The specific enzyme present in the reaction area depends on theparticular analyte for which the electrochemical test strip is designedto detect, where representative enzymes include: glucose oxidase,glucose dehydrogenase, cholesterol esterase, cholesterol oxidase,lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase,lactate oxidase, lactate dehydrogenase, pyruvate oxidase, alcoholoxidase, bilirubin oxidase, uricase, and the like. In many preferredembodiments where the analyte of interest is glucose, the enzymecomponent of the redox reagent system is a glucose oxidizing enzyme,e.g. a glucose oxidase or glucose dehydrogenase.

The second component of the redox reagent system is a mediatorcomponent, which is made up of one or more mediator agents. A variety ofdifferent mediator agents are known in the art and include:ferricyanide, phenazine ethosulphate, phenazine methosulfate,pheylenediamine, 1-methoxy-phenazine methosulfate,2,6-dimethyl-1,4-benzoquinone, 2,5-dichloro-1,4-benzoquinone, ferrocenederivatives, osmium bipyridyl complexes, ruthenium complexes, and thelike. In those embodiments where glucose in the analyte of interest andglucose oxidase or glucose dehydrogenase are the enzyme components,mediators of particular interest are ferricyanide, and the like.

Other reagents that may be present in the reaction area includebuffering agents, e.g. citraconate, citrate, malic, maleic, phosphate,“Good” buffers and the like. Yet other agents that may be presentinclude: divalent cations such as calcium chloride, and magnesiumchloride; pyrroloquinoline quinone; types of surfactants such as Triton,Macol, Tetronic, Silwet, Zonyl, and Pluronic; stabilizing agents such asalbumin, sucrose, trehalose, mannitol, and lactose.

The redox reagent system is generally present in dry form.

CARD AND TEST STRIP MANUFACTURE

The subject electrochemical test strip cards may be fabricated using anyconvenient procedure. In many embodiments, various layers of differentmaterials, e.g., electrode layers, spacer layers, etc., are broughttogether into a single card format, which is then laminated in a barriermaterial to produce the final product. Representative protocols forfabricating different types of cards according to the subject inventionare now described in terms of the figures. However, the followingdescription of representative card manufacture protocols is merelyillustrative, and should in no way be considered limiting, as the cardsmay be fabricated using any convenient protocol, as mentioned above.

FIG. 1 provides a schematic representation of the fabrication of a teststrip card according to one embodiment of the invention. In the processillustrated in FIG. 1, the initial starting materials are top electrodelayer 1 a, bottom electrode layer 1 b and middle spacer layer 1 c. Inthis example, top electrode layer 1 a is a PET substrate with asputtered gold layer on the bottom, while the bottom electrode layer 1 bis a PET substrate with a sputtered palladium layer on the top. Reagents(1 d) are coated onto the bottom layer. Spacer layer is a 3-layerlamination of PSA/PET/PSA (PSA=pressure sensitive adhesive;PET=polyester terepthalate) which has the precursor fluid pathways andreaction chamber present. These three layers are laminated together toproduce structure 2 and a hole 3 is punched through the compositelaminate structure to produce a desiccant chamber. Punching of thedesiccant chamber also results in the production of a fluid stopjunction downstream from the reaction chamber which serves to preciselylimit the amount of fluid sample that enters the strip upon use, asdescribed below. A desiccant material (4), e.g., block, beads, etc., isthen positioned in the punched out desiccant chamber and the resultingstructure is laminated or sealed between top and bottom barrier layers 5a and 5 b consisting of, e.g., PSA-faced aluminum film to produce thefinal card 6. If the layers 5 a and 5 b are sufficiently malleable, thefilm will deform during lamination to allow for the thickness of thedesiccant. If the materials can be embossed, they may be embossed priorto lamination to form a pocket which accepts the desiccant material. Atthe end of card 6 is an information storage means, e.g., barcode,transmitter, etc., which provides information such as calibrationinformation to the meter with which the card is employed. As can beseen, the configuration of the various electrode layers provides forelectrical contacts in the final strips singulated from the cards.

Also shown in FIG. 1 are features cut in the various layers to allowcontacts in the meter to touch the metallized surfaces of the electrodefilms which face the inside of the strip. Additionally, marks (1 e) areshown that indicate lines cut through the metallized layer, but not thebacking material, of the electrode layers, which lines form electricallyisolated areas on the electrode surface. These isolation features servetwo purposes: (1) an electrode is formed at the end of the flow channelwhich allows detection of complete fluid fill of the device, and (2) thearea of the channel actually being used as the electrode is potentiallylimited to areas defined by the features.

An alternate card format that can be produced by the same process isillustrated in FIG. 2. In FIG. 2, the configuration of the initial topand bottom electrode layers has been modified to provide for analternate electrical contact scheme in the electrochemical test stripssingulated from the card. Analogous to the manufacture process depictedin FIG. 1, the first step in the process of FIG. 2 is to provide topelectrode layer 21 a, bottom electrode layer 21 b and middle spacerlayer 21 c. Additionally, marks (21 e) are shown that indicate lines cutthrough the metallized layer, but not the backing material, of theelectrode layers, which lines form electrically isolated areas on theelectrode surface. Reagent material 21 d is present on the surface ofbottom electrode 21 b. The precursors 21 a-21 c are laminated to producestructure 22 and hole is punched out in structure 22 to produce adessicant chamber 23. A desiccant material (24), e.g., block, beads,etc., is then positioned in the punched out desiccant chamber and theresulting structure is laminated or sealed between top and bottombarrier layers 25 a and 25 b consisting of, e.g., PSA-faced aluminumfilm to produce the final card 26.

FIG. 3 provides a schematic illustration of a second protocol that maybe employed to fabricate the subject cards. In the process illustratedin FIG. 3, an initial bottom and spacer layer, 30 b & 30 a,respectively, are employed. The bottom layer 30 b has a metal uppersurface with a reagent stripe (30 c) printed thereon. The bottomelectrode layer has electrode zones defined by isolation cuts 30 d.Middle spacer layer 30 is characterized by having a flow path thatincludes a desiccant chamber, where the desiccant chamber 33 is incommunication with the reaction chamber in the adjacent strip precursor.The bottom and middle layers are first laminated together to producestructure 32, and desiccant material 37 is positioned in the desiccantchamber 33. Structure 32 is then laminated to top electrode layer 34 a(which has electrode isolation cuts 34 b) to produce final card 35. Inthe embodiment shown in FIG. 3, the electrode film serves two functions:(a) support for the metal layer and (b) as a primary moisture barrier.In this embodiment, the film is composed of a material with low moisturevapor transmission rates, such as the Aclar material available fromAllied Signal. A pocket is pre-formed in the film, e.g., by stamping orthermoforming, to accept the desiccant material. The outer barrier ofthe film is directly adjacent to the spacer layer, so a stop junctioncannot be formed by the desiccant chamber. Therefore, the desiccantchamber is positioned on the adjacent strip, such that a stop junctionis produced upon singulation of the card into strips.

To produce electrochemical test strips from the cards, the cards aresingulated or cut into the test strips. Any convenient cutting orseparation protocol may be employed, including slitting, shearing,punching, laser singulation, etc. In certain embodiments, singulation isperformed by the meter with which the strip is employed.

METHODS OF USE

In using the electrochemical test strips produced from the subjectcards, a quantity of the physiological sample of interest is introducedinto the electrochemical cell of the reaction chamber of the test strip.The physiological sample may vary, but in many embodiments is generallywhole blood or a derivative or fraction thereof, where whole blood is ofparticular interest in many embodiments. The amount of physiologicalsample, e.g., blood, that is introduced into the reaction area of thetest strip varies, but generally ranges from about 0.1 to 10 μL, usuallyfrom about 0.9 to 1.6 μL. The sample is introduced into the reactionarea using any convenient protocol, where the sample may be injectedinto the reaction area, allowed to wick into the reaction area, and thelike, as may be convenient.

Following application of the sample to the reaction zone, anelectrochemical measurement is made using the reference and workingelectrodes. The electrochemical measurement that is made may varydepending on the particular nature of the assay and the device withwhich the electrochemical test strip is employed, e.g. depending onwhether the assay is coulometric, amperometric or potentiometric.Generally, the electrochemical measure will measure charge(coulometric), current (amperometric) or potential (potentiometric),usually over a given period of time following sample introduction intothe reaction area. Methods for making the above describedelectrochemical measurement are further described in U.S. Pat. Nos.:4,224,125; 4,545,382; and 5,266,179; as well as WO 97/18465; WO99/49307; the disclosures of which are herein incorporated by reference.

Following detection of the electrochemical signal generated in thereaction zone as described above, the amount of the analyte present inthe sample introduced into the reaction zone is then determined byrelating the electrochemical signal to the amount of analyte in thesample. In making this derivation, the measured electrochemical signalis typically compared to the signal generated from a series ofpreviously obtained control or standard values, and determined from thiscomparison. In many embodiments, the electrochemical signal measurementsteps and analyte concentration derivation steps, as described above,are performed automatically by a devices designed to work with the teststrip to produce a value of analyte concentration in a sample applied tothe test strip. A representative reading device for automaticallypracticing these steps, such that user need only apply sample to thereaction zone and then read the final analyte concentration result fromthe device, is further described in copending U.S. patent applicationSer. No. 6,193,873.

The methods may be employed to determine the concentration of a varietyof different analytes, where representative analytes include glucose,cholesterol, lactate, alcohol, and the like. In many preferredembodiments, the subject methods are employed to determine the glucoseconcentration in a physiological sample. While in principle the subjectmethods may be used to determine the concentration of an analyte in avariety of different physiological samples, such as urine, tears,saliva, and the like, they are particularly suited for use indetermining the concentration of an analyte in blood or blood fractions,e.g., blood derived samples, and more particularly in whole blood.

KITS

Also provided by the subject invention are kits for use in practicingthe subject invention. The kits of the subject invention at leastinclude an electrochemical test strip, as described above. The subjectkits may further include a means for obtaining a physiological sample.For example, where the physiological sample is blood, the subject kitsmay further include a means for obtaining a blood sample, such as alance for sticking a finger, a lance actuation means, and the like. Inaddition, the subject kits may include a control solution or standard,e.g., a glucose control solution that contains a standardizedconcentration of glucose. Finally, the kits include instructions forusing the subject reagent test strip cards in the determination of ananalyte concentration in a physiological sample. These instructions maybe present on one or more of the packaging, a label insert, containerspresent in the kits, and the like. Alternatively, a means for remotelyaccessing such instructions, e.g., at an internet site, may be provided,where such means may take the form of a URL printed onto a substratepresent in the kit, e.g., package insert, packaging etc.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example I

Palladium and gold coated polyester films were treated withmercapto-ethane sulfonic acid (MESA) by dipping in a 0.6 M MESAsolution, followed by air drying. The palladium foil was laminated to aspacer layer with the channel shape shown in FIG. 4 a. Apyrrolo-quinoline-quinone (PQQ)—glucose dehydrogenase (GDH) reagent wasformulated as follows:

Solution A

1.1 g CaCl₂+100 mL deionized water

Solution B

99.5 mL (0.1 M Citracconic acid, pH 6.5, 0.02% Silwet 7600)+0.5 mL A

Solution C

1 mg PQQ+27.5 mL B

Solution D

1.12 g K₄Fe(CN)₆+5 mL B

Solution E

3.21 mg GDH (502 U/mg)+300 μL C

incubate for 30 minutes at RT in the dark

add 100 μL D

1.5 μL of the reagent was applied with a pipet to the reagent zone (Ifin FIG. 1), and air dried on a 50° C. hot plate. The gold coated filmwas applied to the top of the spacer layer, and the punched desiccantchamber was created as shown in FIG. 1. At this point, cards werefinished by either inserting three 4A mol sieve beads (approx. 2 mgeach) and covering with aluminum foil (3M 425), or laminating anotherlayer of 0.005″ gold-coated polyester film to cover the punched hole.The three mol sieve beads together had a total capacity of about 1.2 mgof water.

The cards were stored for 32 days either in a 75% RH, room temperaturechamber, or desiccated (4A mol sieve) in at 5° C. At intervals duringthe study, cards were removed, strips were singulated and then developedwith 42% hematocrit blood adjusted to approximately 0, 40 and 450 mg/dlglucose. A different donor's blood was employed at each time point, butthe refrigerated control was included for comparison in the case of anydonor-related effects (and differences in actual glucose levels). 6-9strips were developed for each case.

The device which read the strips applied a +50 mV potential across theelectrodes to detect sample application. When a current increasesignaled sample application, the potential was changed to −300 mV andheld there for 5 seconds. After 5 seconds, the potential was changed to+300 mV and held there for 9 seconds. During the +300 mV phase, thedecaying current vs time curve was projected mathematically to infinity;this infinity current value was termed i_(SS). I_(SS) is approximatelyproportional to glucose concentration. FIGS. 5 a, 5 b and 5 c show theaveraged i_(SS) values for the two cases and the refrigerated control.At zero glucose, a small (about 10 microvolt) background current is seeninitially for all cases. This current remains essentially unchanged forall cases except the PET case exposed to high humidity, where itincreased dramatically as the study progressed, indicating a build-up offerrocyanide. At 40 mg/dl glucose, the effect was essentially the same.At 450 glucose, where the glucose-related current was much higher, theincrease in current due to ferrocyanide production on exposure was notas noticeable as a decrease in i_(SS) due to enzyme degradation. Again,this degradation effect occurred only in the PET high humidity case.Clearly, the foil-faced internally desiccated strips were far morestable when challenged with this high humidity environment for up to 32days.

Example II

In this example, cards similar to the aluminum-faced cards in example Iwere prepared, with one exception (see FIGS. 6 a and 6 b). The sampleentrance and vent channels were shortened so that when strips weresingulated, the channel system was still completely sealed inside thestrip, and the tips of the sample and vent channels ended 0.030″ fromthe edge of the strip (this merely involved shortening the channels by0.060″). This configuration was intended to simulate a cardconfiguration in which cuts are made between strips at time ofmanufacture to minimize the force required for singulation, as outlinedabove. Samples were prepared both as complete, uncut cards, and assingulated strips. Each configuration was also prepared with and withoutdesiccant in the desiccant chamber.

To investigate the effect of the cuts on moisture ingression, and tocorrelate the previously observed card stability with moisture insidethe package, a moisture uptake study was conducted as follows: 40individual strips were prepared for each singulated strip case, and 220-strip cards were prepared for the card cases. All four cases wereplaced in the 75% RH, room temperature chamber. Over the next 63 days,all materials in each case were weighed to assess moisture uptake. Tocompute the amount of moisture passing through the strip or cardpackage, the weight gain of the non-desiccant case was subtracted fromthat of its corresponding desiccant-containing configuration. Based onthe observation that each of the 3 beads per strip weighed about 2 mgand could absorb about 20% of its weight in moisture, the percentexhaustion of the desiccant was calculated at each time point. FIG. 7shows the results.

The complete card configuration had slightly more than 40% exhaustion ofthe desiccant in 63 days. In example I, the good reagent stability seenup to 32 days with complete cards, in retrospect, corresponds to about18% exhaustion of the desiccant. Because mol sieve maintains very lowrelative humidity even at significant degrees of exhaustion, one wouldgood reagent stability to be found up to 40% exhaustion as well.

The singulated strip configuration, on the other hand, reached 50%exhaustion in about 5 days; significantly faster than the complete card.The singulation cut opens up routes which speed up moisture ingression.Thus with this foil-faced configuration, the meter would probably haveto make the entire cut between strips. Also, the life of the end strip(and possibly the next one or so) might be less than interior strips.

Example III

See FIGS. 8 a and 8 b. In this example, cards were made as in exampleII, except that (1) the rectangular shape of the desiccant chamber wascut into the center spacer layer, (2) metallized PET and the foil outerlayers were replaced with a single layer of 0.005″ Aclar 22C, (3) a0.028″ pocket was formed (by cold stamping) in one layer of the Aclar toconform to the shape of the desiccant chamber and (4) a 13 mg piece mgpiece of 0.025″ desiccant tape consisting of about 60% mol sieve powderand 1-3% glycol in PETG (Capital vial) was used as desiccant. Thedesiccant had a total capacity of about 2.6 mg of water per strip, orabout 2.3× the capacity of the 3 mol sieve beads in examples I and II.For comparison, foil faced cards were made as in I and II, but the molsieve beads were replaced with the same amount of desiccant tape as theAclar cards (see FIGS. 9 a and 9 b). Both types of cards were also madewithout desiccant as a control for moisture absorption by the outside ofthe package, and all configurations were subjected to 75% RH as cardsand cut strips. 40 strips were tested per case

FIGS. 10 a and 10 b show the results. The foil singulated strips in thisexample exhibited much better moisture resistance than in example II:the 50% exhaustion level was reached at about 12 days rather than in 5days; this is approximately what would have been predicted from theincreased desiccant capacity.

The Aclar 22C data shows an anomalous exhaustion decrease between day 0and day 1; this is undoubtedly a weighing error at day 0. After allowingfor this offset (all exhaustion values should be about 5-10% higher), itis clear that at 28 days, the cut strips have not gained enough moistureto exhaust the desiccant more than 35%, and that the desiccant shouldcertainly be less than 50% exhausted at 30 days. Thus the change inmaterials and amount of desiccant have both contributed to achieving adesign where even if pre-singulation cuts are made between strips, thestrips should remain dry enough to last at least one month, and endstrips should be just as good as center strips.

The Aclar strip used in this example is intended to be a model formoisture vapor transmission through a package similar to the intendeddevice.

The above results and discussion demonstrate that improvements inelectrochemical test strip technology are provided by the subjectinvention. Specifically, the subject invention provides for storagestable multi-strip cards or tapes that can be singulated as needed bythe end user, which will provide for less use of packaging materials andmore efficient and cost effective manufacture protocols, among otheradvantages. As such, the subject invention represents a significantcontribution to the art.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. The citation of any publication is for its disclosure priorto the filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A method of fabricating an electrochemical test strip card comprisinga plurality of test strips configured to be singulated from said teststrip card, said method comprising: providing a top layer, a bottomlayer and a spacer layer therebetween, wherein said spacer layercomprises a pattern for each of said plurality of test strips thatprovides for at least a fluid ingress channel, a reaction chamber, adesiccant chamber, a channel interconnecting said reaction chamber andsaid a desiccant chamber, and a venting channel; positioning a desiccantmaterial in said desiccant chamber; and laminating together said toplayer, said bottom layer and said spacer layer.
 2. The method of claim 1further comprising punching a hole in at least said spacer layer to formsaid desiccant chamber.
 3. The method of claim 1 further comprisingpunching a hole in said top layer, said bottom layer and said spacerlayer to form said dessicant chamber.
 4. The method of claim 1 whereinsaid top layer comprises a top surface, a bottom surface and anelectrode on said bottom surface and wherein said bottom layer comprisesa top surface, a bottom surface and an electrode on said top surface. 5.The method of claim 4 wherein said top layer further comprises a barrierlayer on said top surface and wherein said bottom layer furthercomprises a barrier layer on said bottom surface.
 6. The method of claim1 wherein said desiccant material comprises one or more beads.
 7. Themethod of claim 1 wherein said desiccant material comprises a block. 8.The method of claim 1, further comprising forming a singulation precutin said laminated structure.
 9. The method of claim 1, furthercomprising singulating each of said plurality of test strips from saidtest strip card.
 10. The method of claim 1, wherein said test strip cardcomprises an information storage means for storing information to beused by a test strip meter with which said test strip card is to beused.
 11. The method of claim 10, wherein said information comprisescalibration information.
 12. The method of claim 11, wherein saidinformation storage means comprises a barcode.
 13. The method of claim1, wherein said test strip card comprises electrical contacts for usewith a test strip meter.
 14. A method of frabricating an electrochemicaltest strip card comprising a plurality of test strips configured to besingulated from said test strip card, said method comprising: providinga top electrode layer, a bottom electrode layer and a spacer layertherebetween, wherein said spacer layer comprises a pattern thatprovides for at least a fluid ingress channel, a reaction chamber, alocation for a desiccant chamber, a channel interconnecting saidreaction chamber and said location for a desiccant chamber, and aventing channel; laminating together said top layer, said bottom layerand said spacer layer; punching a hole through said laminated structureat said desiccant chamber location thereby forming said desiccantchamber; positioning a desiccant material in said desiccant chamber; andproviding a first barrier layer on top of said laminated structure and asecond barrier layer on the bottom of said laminated structure whereinsaid descant chamber is in gaseous communication with said reactionchamber.
 15. The method of claim 14, further comprising laminating saidfirst and second barriers to said previously laminated structure. 16.The method of claim 14, wherein said top electrode layer comprises apolyester terepthalate substrate having a top surface and a bottomsurface and a gold layer on said bottom and wherein said bottomelectrode layer comprises a polyester terepthalate substrate having atop surface and a bottom surface and a palladium layer on said topsurface and a reagent system on said palladium layer.
 17. The method ofclaim 14 wherein said spacer layer comprises a layer of polyesterterepthalate having a top surface and a bottom surface, and a pressuresensitive adhesive on said top surface and said bottom surface.
 18. Themethod of claim 14, wherein the punching of the desiccant chambercomprises forming a fluid stop junction downstream from the reactionchamber.